PROJECT SUMMARY This application for renewal seeks to better understand the neurobiological mechanisms of nicotine addiction. A major accomplishment during the previous funding cycle was identifying the critical role played by the habenula-interpeduncular nucleus (habenula-IPn) circuit in regulating the motivational properties of nicotine. We also found that the neuropeptide glucagon-like peptide-1 (GLP-1), released from neurons that arise in the nucleus of the solidary tract (NTS), enhances the activity of the habenula-IPn circuit and thereby inhibits nicotine intake. Transcription factor 7-like 2 (Tcf7l2) is considered a core component of the GLP-1 signaling cascade. In exciting new preliminary data, we find that Tcf7l2 is highly enriched in the habenula-IPn circuit. Using a new line of Tcf7l2 knockout (Tcf7l2-/-) rats, we also find that disruption of Tcf7l2 signaling dramatically increases the motivation to consume nicotine. RNAi-mediated knockdown of Tcf7l2 in the habenula of wild- type rats similarly increases nicotine intake. These exciting new findings suggest that habenular Tcf7l2 signaling plays a critical role in regulating the motivational properties of nicotine. In this competitive renewal, we will use cutting-edge molecular, cellular and behavioral approaches to investigate the mechanisms of Tcf7l2 action. In AIM I, we will investigate cellular mechanisms of Tcf7l2 action. First, we will confirm that virus- mediated re-expression of Tcf7l2 specifically in the habenula of Tcf7l2-/- rats rescues their otherwise increased motivation to consume nicotine. Second, we will investigate the role of Tcf7l2 in regulating the responsiveness of habenular neurons to self-administered nicotine. This will be accomplished by expressing the genetically encoded calcium indicator GCaMP6f in the habenula of wild-type and Tcf7l2-/- rats and using in vivo fiber photometry to quantify calcium transients evoked by self-administered nicotine. Third, we will identify the precise population of habenular neurons in which Tcf7l2 acts to control nicotine intake. This will be accomplished by using in vivo CRISPR to delete Tcf7l2 in genetically defined populations of habenular neurons of mice and characterizing their nicotine intake. In AIM II, we will identify transcriptional mechanisms by which Tcf7l2 acts in habenula to control nicotine intake. We will use chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and RNA-Seq on habenula tissues from wild-type and Tcf7l2-/- rats to identify habenular genes directly regulated by Tcf7l2. In AIM III, we will identify molecular mechanisms by which Tcf7l2 controls nicotine intake. We will use in vivo CRISPR to delete the most promising Tcf7l2 target genes in habenula, or perturb signaling networks in which target genes are enriched, and assess the impact on nicotine intake. This highly innovative renewal builds logically on the substantial progress made during the previous cycle and promises to yield significant new insights into the mechanisms of nicotine addiction.